JP2965682B2 - New peptides, their production methods and applications - Google Patents

Description

The present invention provides a novel peptide having the following structure, and relates to a peptide useful as an angiotensin converting enzyme inhibitor or the like.

Phe-Phe-Gly-Arg-Cys-Val-Ser-Pro [Prior art] Angiotensin converting enzyme is mainly present in lung, vascular endothelial cells, and renal proximal tubules, and angiotensin I (As
Acts on p-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) to release the dipeptide (His ⁹ -Leu ¹⁰ ) from the C-terminus of angiotensin I and has a strong pressor action It is an enzyme that produces angiotensin II. This enzyme also has the action of destroying and inactivating bradykinin, which is a hypotensive substance in the living body, and is strongly involved in the pressor system.

Hitherto, it has been considered that inhibiting the activity of an angiotensin converting enzyme acts on blood pressure lowering, and is clinically effective for preventing and treating hypertension.

Recently, since captopril, a proline derivative, has been synthesized and its antihypertensive activity has been confirmed, studies on the synthesis of various angiotensin converting enzyme inhibitors have been actively conducted, and attempts are being made to obtain them from natural products.

This is because a natural product-derived angiotensin converting enzyme inhibitor is expected to be a low-toxicity and highly safe antihypertensive agent because it is obtained from foods or food raw materials.

[Problems to be Solved by the Invention] However, angiotensin converting enzyme inhibitors found in natural products are extremely rare, such as teprotide (nonapeptide, SQ20881) slightly obtained from Brazilian or Japanese snake venom, and Streptomyces. Only the metabolite IC83 of actinomycetes belonging to the genus (JP-A-58-177920) is known. Also, as angiotensin converting enzyme inhibitors obtained by enzymatic treatment of natural products, peptides and the like obtained by decomposing milk casein with trypsin are known (JP-A-58-109425, JP-A-58-109425). −44323,
No. 59-44324, No. 61-36226, No. 61-36227) The development of new inhibitors is being demanded.

[Means for Solving the Problems] The present inventors have intensively searched for an angiotensin converting enzyme inhibitor which is a natural substance and has few side effects in order to solve such problems, and as a result, a composition obtained by hydrolyzing albumin with a specific enzyme was used. The presence of a substance having angiotensin-converting enzyme inhibitory activity, the substance being identified as Phe-Phe-Gly-Arg-
Cys-Val-Ser-Pro was found to be a peptide,
The present invention has been completed.

The peptide Phe-Phe-Gly-Arg-Cys-Val-Ser-Pro of the present invention is a novel peptide not described in the literature, and is produced by hydrolyzing albumin with pepsin. It may be used, or may be used after purification if necessary. Furthermore, it can also be produced by synthesizing by applying conventional means of peptide synthesis.

Phe is phenylalanine, Gly is glycine,
Arg is arginine, Cys is cysteine, Val is valine, Ser
Represents serine and Pro represents proline, and all such amino acids are in L-form.

The peptide of the present invention can be obtained by hydrolyzing albumin with pepsin or by peptide synthesis.
In order to hydrolyze albumin with pepsin, the formulation differs depending on the properties of albumin, but in the case of poor solubility, mix albumin in hot water, homogenize with strong stirring, add a predetermined amount of pepsin and add a temperature of 10-60 ° C. , Preferably 20-40
The mixture is allowed to stand or stirred at 10 ° C. and a pH of 0.1 to 4.0 for 10 minutes to 3 days. As albumin, soluble proteins widely distributed in body fluids and tissues of animals and plants, for example, ovalbumin, serum albumin, milk albumin, muscle albumin and the like are optionally used, and ovalbumin is particularly useful. In the hydrolyzate, other peptides are present in addition to the peptide of the present invention.These peptides may be used for various purposes as a mixture, or only the peptide of the present invention may be isolated and used. No problem.

In the case of isolation, the hydrolyzed solution is filtered by a known operation such as centrifugation. After the application of extraction, concentration, drying, etc., with or without application, differences in adsorption affinity for various adsorbents, differences in solubility or solubility in various solvents, two immiscible liquid phases It is preferable to isolate the target compound by applying a means utilizing a difference in distribution between molecules, a difference in elution rate based on the size of a molecule, a property of precipitation from a solution or a difference in deposition rate. These methods may be used alone as needed or combined in any order and applied repeatedly.

The peptide of the present invention is a raw material having a reactive carboxyl group corresponding to one of two fragments bisected at an arbitrary position of a peptide bond by a method usually used for peptide synthesis, that is, a liquid phase method or a solid phase method, It is also produced by condensing a raw material having a reactive amino group corresponding to the other fragment using a carbodiimide method, an active ester method, or the like, and, if the condensate formed has a protective group, removing the protective group. obtain.

Functional groups that should not take part in the reaction in this reaction step are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl, t-butyloxycarbonyl, p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and the like. Examples of the carboxyl-protecting group include groups capable of forming an alkyl ester, a benzyl ester and the like. In the case of the solid phase method, the C-terminal carboxyl group is a chloromethyl resin, an oxymethyl resin, a P-alkoxybenzyl alcohol resin. And the like.

The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.

After completion of the condensation reaction, the protecting group is removed, but in the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved.

Further, the peptide of the present invention is purified according to a usual method. For example, ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like can be mentioned.

The administration route of the peptide used in the present invention may be any of oral administration, parenteral administration and rectal administration, but oral administration is preferred. The dose of the peptide of the present invention varies depending on the type of the compound, the administration method, the symptoms and age of the patient, etc., but is usually 0.01 to 1000 mg, preferably 0.01 to 10 mg once.
1-3 times per day. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a preparation carrier. As the pharmaceutical carrier, a substance which is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium, ion exchange resin, methylcellulose , Gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone,
Polyvinyl alcohol, light silicic anhydride, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, Wax, liquid paraffin,
Examples include white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water and the like. Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another appropriate medium at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the peptide of the present invention is prepared by dissolving the peptide in water, but may be dissolved in a physiological saline solution or a glucose solution if necessary, or may be added with a buffer or a preservative. Good.

These preparations can contain the peptide of the present invention at a rate of 0.01% or more, preferably 0.5 to 70%. These formulations may also contain other therapeutically valuable components.

[Action] The peptide of the present invention is a novel peptide and has an excellent angiotensin converting enzyme inhibitory action, exhibits a blood pressure lowering action, a bradykinin inactivation inhibitory action, and has essential hypertension, renal hypertension, adrenal hypertension, Prevention of hypertension,
It is useful as a therapeutic agent, a diagnostic agent for these diseases, an antihypertensive agent used in various disease states, an increase in the threshold of angina attack, a decrease in myocardial infarction, and an improvement agent for disease states in congestive heart failure.

Example Reference Example Next, the present invention will be described more specifically with reference to examples. After diluting and dissolving the raw egg white five-fold with distilled water, PH-1.6 in IN-HCl.
Pepsin in lysate (containing 20mg / ml protein) adjusted to
0.2 mg / ml (manufactured by Sigma) was added, and the reaction was allowed to stand at 37 ° C. for 3 hours. The reaction was stopped by boiling at 100 ° C. for 10 minutes. The reaction solution was centrifuged at 10,000 rpm for 5 minutes, concentrated, and purified by high performance liquid chromatography (ODS-, PH- and CN-column) to obtain a peptide.

The amino acid sequence of this product was analyzed by an automatic Edman degradation method using a gas phase protein sequencer (Model 477A, manufactured by Abride Biosystems) to obtain the following structure.

H-Val-Ser-Pro-OH The physical properties of the peptide are as follows.

TLC [n-butanol: acetic acid: pyridine: water = 15: 3: 10: 1
2] (silica gel plate, ninhydrin coloring) Rf: 0.43 mp: 91 ° C Elemental analysis C ₁₃ N ₂₃ N ₃ O ₅ · 0.5H ₂ O CH N calculated 50.31 7.79 13.54 measured 50.19 7.69 13.52 Specific rotation ▲ [ α] ²⁵ _D ▼; (C = 0.5 water); −81.37 Synthesis of peptide (peptide synthesis of the present invention) Commercially available Boc (butoxycarbonyl) -Pro-O-Resin
0.83 g was collected in a reaction vessel of a peptide synthesizer SAM2 manufactured by Biosearch and synthesized as follows.

After removing the Boc group by reaction in methylene chloride containing 45% trifluoroacetic acid and 2.5% anisole for 25 minutes, washing with methylene chloride, neutralization with methylene chloride containing 10% diisopropylethylamine, and washing with methylene chloride Was done.

This was mixed with 5 ml of a 0.4 M solution of 0.4 M Boc-Ser (Bzl) (benzyl group) in dimethylformamide and 5 ml of a 0.4 M solution of 0.4 M diisopropylcarbodiimide in methylene chloride. Was.

The obtained resin was washed with dimethylformamide, methylene chloride, a mixed solution of methylene chloride containing 10% diisopropylethylamine, methylene chloride, and a mixture of methylene chloride and dimethylformamide to obtain a Boc-Ser (Bzl) -Pro resin. Subsequently, the same removal of the Boc group and the coupling of the Boc and the amino acid were repeated to repeat Phe-Phe-Gly-Arg-Cys-Val.
-Ser (Bzl) -Pro resin was obtained.

The resin was stirred in 20 ml of hydrogen fluoride containing 10% anisole at 0 ° C. for 1 hour to release the peptide from the resin.
Hydrogen fluoride was distilled off under reduced pressure, the residue was extracted with 30% acetic acid, and lyophilized to obtain a crude peptide. Use this with an ODS column (Cosmo
sil 5C ₁₈₎ and purified by reverse phase chromatography on, H-Phe-Phe-Gly -Arg-Cys-Val-Ser-Pro-OH
(Yield 100 mg) was obtained.

The product was analyzed using the same protein sequencer as described above, and as a result, the product was found to have the above composition.

 The physical properties of the peptide are as follows.

TLC Rf: 0.60 mp: 165 ℃ Elemental analysis C ₄₅ H ₆₆ N ₁₂ O ₁₂ O ₂・ 0.8H ₂ O CH N S Calculated 51.69 6.52 16.08 6.13 Measured 51.60 6.48 16.00 6.18 Specific rotation ▲ [α] ²⁵ _D ▼; (C = 0.5 water); −82.63 The peptide of the present invention can be obtained by the same method as in the Reference Example, and can be fractionated from the product solution after hydrolysis in the Reference Example.

Example 1 (Measurement of angiotensin converting enzyme inhibitory activity) The measurement of angiotensin converting enzyme inhibitory activity was performed using Cheung
And Cushman's method (Biochemical Pharamacology 20 , 1637
(1971)] according to the following method.

Enzyme substrate: Bz (benzyl) -Gly-His-Leu (86 mg water
8 ml and a solution dissolved in 8 ml of phosphate buffer) Enzyme; rabbit lung acetone powder (manufactured by Sigma) (1 g is crushed in 10 ml of 50 mM phosphate buffer and centrifuged, and the supernatant is centrifuged) 100μ of the enzyme substrate, 12μ of the enzyme solution and the peptide of the present invention at a predetermined concentration were mixed, and the whole was mixed with water to 250μ.
After that, the reaction was carried out at 37 ° C. for 30 minutes.

The reaction was terminated using 250 μl of 1N-NCl. After completion of the reaction, 1.5 ml of ethyl acetate was added, the mixture was stirred with Vortex for 15 seconds, and the mixture was centrifuged.

1.0 ml was taken out from the ethyl acetate layer, ethyl acetate was distilled off, 1 ml of distilled water was added to dissolve the residue, and the value of the ultraviolet absorption ₂₂₈ nm (OD ₂₂₈ ) of the extracted hippuric acid was measured.

Percent inhibition by the OD ₂₂₈ of when reacted without inhibitor as 100%, the concentration IC of the inhibitor when the OD ₂₂₈ of the inhibition rate of 50% determined as 0% when the reaction time of 0 minutes (the peptide of the present invention)
_The activity was indicated at ₅₀ (μM).

 The results are shown in Table 1.

[Effect] In the present invention, a novel peptide useful as an angiotensin converting enzyme inhibitor can be obtained.

────────────────────────────────────────────────── ─── Continuation of front page (56) References JP-A-61-87886 (JP, A) Chemical Abstracts Vol. 81, No. 34218 & Hypertension [Proc. Symp. Meeting Date 1971, P541-547 (1972) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 7/08, 5/083, 14/76 A61K 38/00 CA (STN) REGISTRY (STN )

Claims (4)

(57) [Claims] (1) Phe-Phe-Gly-Arg-Cys-Val-Ser-Pro
New peptide. 2. Phe-Phe-Gly-Arg-Cys-Val-Ser-Pro, characterized in that albumin is hydrolyzed with pepsin.
A method for producing a novel peptide. 3. The method according to claim 2, wherein ovalbumin is used as the albumin. 4. Phe-Phe-Gly-Arg-Cys-Val-Ser-Pro
Angiotensin converting enzyme inhibitor comprising a peptide as an active ingredient.